There are currently more than 18 Helicobacter species described and the numbers are increasing. Many of these bacteria are not culturable by standard laboratory methods, because they require microaerophilic conditions and also the presence of hydrogen gas for growth. Many laboratories classify gram-negative, urease-positive spiral bacteria isolated from gastric specimens as H. pylori without further work-up. Consequently, the incidence and pathogenicity of Helicobacter spp. other than H. pylori are relatively unclear. Nonculturable Helicobacter-like organisms have been reported in human gastric biopsies and there are reports of patients with a positive urease breath test with negative gastric biopsy cultures for H. pylori. It is, at present, unknown whether or not these cases are due to a Helicobacter spp. other than H. pylori. There are several reports of Helicobacter-like organisms in blood. Most Helicobacter spp. are closely related by 16S rRNA sequencing and possess a rapid urease activity similar to H. pylori. The urease enzyme is strongly expressed and has been considered as a target for vaccine development against H. pylori. However, the database of Helicobacter spp urease DNA sequences is limited to three species other than H. pylori. Using polymerase chain reaction (PCR) with degenerate primers, we were able to amplify a region of the ureB gene from H. hepaticus and F. rappini. These PCR products were cloned and sequenced. Analysis of these sequences revealed considerable variability when compared with available sequences. The ureB gene should prove to be a useful PCR amplification target for identification and differentiation between fastidious or nonculturable Helicobacter spp. We have also improved our PCR assay for rapid identification of fastidious Helico-bacter-like organisms in patient specimens. We have identified two F. rappini isolates in blood using PCR of the urease gene. This information should be useful in studies of the prevalence and clinical significance of Helicobacter species other than H. pylori. The results of this project were presented as a poster at the annual Molecular Diag-nostics and Therapeutics meeting of the American Society for Microbiology. This project is completed.